An increase in plasma adiponectin multimeric complexes follows hypocaloric diet-induced weight loss in obese and overweight premenopausal women. Clin Sci (Lond

A B S T R A C T Adiponectin is involved in the regulation of glucose and fatty acid metabolism, influences wholebody insulin sensitivity and protects arterial walls against the development of atherosclerosis. Plasma adiponectin is decreased in obese, insulin-resistant and Type 2 diabetic patients. Adiponectin circulates in plasma as high-, medium-and low-molecular-weight ('mass') forms (HMW, MMW and LMW respectively). The HMW form is believed to be closely associated with insulin sensitivity. The aim of the present study was to investigate whether diet-induced changes in body weight and insulin sensitivity were associated with changes in the quantity of adiponectin multimeric complexes. A total of 20 overweight or obese women (age, 39.4 + − 9.5 years; body mass index, 32.2 + − 6.4 kg/m 2 ) underwent 12 weeks of low caloric diet (600 kcal/day less than energy requirements; where 1 kcal ≈ 4.184 kJ). Plasma samples were drawn before and after the study for biochemical analysis and Western blot detection of adiponectin multimeric complexes. The hypocaloric diet resulted in a weight reduction (89.8 + − 16.4 kg compared with 83.1 + − 15.6 kg; P < 0.001) and an improvement in whole-body insulin sensitivity, as measured by HOMA (homoeostasis model assessment index; 1.9 + − 0.8 compared with 1.5 + − 0.7; P = 0.013). Increases in the quantities of the HMW, MMW and LMW forms by 5.5, 8.5 and 18.1 % respectively, were observed (P < 0.05 for all of the forms). Total plasma adiponectin was increased by 36 % with borderline significance (P = 0.08). No correlations between changes in adiponectin complexes and changes in indices of insulin sensitivity were observed. In conclusion, diet-induced weight loss improved insulin sensitivity as well as increased the amount of HMW, MMW and LMW adiponectin complexes in plasma

Structure et fonction d'une cytokinine oxydase / déshydrogénase (modifications d'enzymes)

L'objet de cette thèse est l'étude d'une cytokinine oxydase/déshydrogénase du maïs (ZmCKO1). La flavoenzyme CKO catalyse la dégradation des hormones végétales de type cytokinines, dérivés N6-substitués de l'adénine. Il s'agissait de déterminer l'importance du rôle de CKO dans le développement des plantes, la structure de l'enzyme et l'acide aminé clef dans la réaction enzymatique, le mécanisme réactionnel en présence de deux types d'inhibiteurs différents, la localisation des sites catalytiques oxydase et déshydrogénase?Afin de mieux comprendre le rôle de la CKO dans l'homéostasie des cytokinines, la teneur en hormone a été modifiée en surexprimant ZmCKO1 de manière constitutive chez Arabidopsis thaliana. Les effets phénotypiques et les teneurs en cytokinines ont été déterminés de même que l'expression de gènes endogènes impliqués dans la biosynthèse, la dégradation et la signalisation des cytokinines. ZmCKO1 a été exprimée dans la levure Yarrowia lipolytica, purifiée et ses propriétés moléulaires et cinétiques caractérisées ; elle a été cristallisée et sa structure obtenue avec une résolution de 1,95 Å en présence de deux types d'inhibiteurs, des analogues d'adénine N6-substituée et de cytokinines de type urée. Des expériences de mutagenèse dirigée confirment le rôle clef de Asp169 dans la catalyse enzymatique et indiquent l'importance d'autres résidus d'acide aminé dans l'interaction avec le substrat et les accepteurs d'électrons. Un mécanisme réactionnel de type ping-pong est proposé, la molécule organique acceptrice d'électrons, tout comme l'oxygène, se liant au site actif en face de l'azote N5 du FAD.The Ph.D. thesis is focused on the study of cytokinin oxidase/dehydrogenase from maize (ZmCKO1). CKO is a flavoenzyme and catalyzes the degradation of plant hormones cytokinins, which are N6-substituted adenine derivatives. Four questions were adressed: (i) how important is the role of CKO in plant development? (ii) what is the structure of the enzyme and what is the key amino acid residue in enzyme reaction? (iii) what is the mechanism of CKO inhibition with two different types of synthetic cytokinin derivatives and whether is the inhibition accompanied by enzyme modification? (iv) are the oxidase and dehydrogenase catalytic sites different?To unravel the role of CKO in cytokinin homeostasis, content of the hormone was altered by constitutive overexpression of ZmCKO1 in Arabidopsis thaliana. Effects on phenotype and changes in levels of cytokinins were determined as well as expression of endogenous genes related to cytokinin biosynthesis, degradation and signaling. ZmCKO1 was further expressed in the yeast Yarrowia lipolytica, purified, and its molecular and kinetic properties were characterized; it was further crystallized and its structure solved up to 1.95 Å resolution in presence of two types of inhibitors - N6-substituted adenine and urea-type cytokinin analogs. Site-directed mutagenesis confirmed the key role of Asp169 in enzyme catalysis and revealed the importance of some other amino acid residues in the interaction with substrate and electron acceptors. This work leads to the proposal that the CKO reaction proceeds through a pingpong mechanism and that organic electron acceptor as well as oxygen bind in the active site in front of nitrogen N5 of FAD.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Aminoaldehyde dehydrogenase activity during wound healing of mechanically injured pea seedlings.

International audienceAminoaldehyde dehydrogenase (AMADH, EC 1.2.1.19) is an enzyme that, in association with amine oxidase, participates in polyamine catabolism. In plants, the enzyme is well characterized in pea seedlings. In this study, we used etiolated and light-grown pea seedlings as model plants to evaluate the possible AMADH role in response to stress caused by mechanical damage. In the beginning, the activity distribution of AMADH, amine oxidase and peroxidase in organs of 7-day-old intact pea seedlings was analyzed. To perform mechanical damage, stems of 10-day-old seedlings were each divided into four segments of equal length. The top (=fourth) segments were then longitudinally cut with a lancet. During healing, the injured segments and their control counterparts were harvested in 1-day intervals and analyzed for activity of the above enzymes, polyamine and 4-aminobutyrate (GABA) concentrations. The injury elicited increases in AMADH, amine oxidase and peroxidase activities in both etiolated and green seedlings, accompanied by parallel increases in putrescine, cadaverine, spermidine and GABA content. Histochemical experiments allowed visualization of increased AMADH activity in cross sections obtained from the injured stem segments. The activity was localized in cortical parenchyma and epidermal cells adjacent to the wound site in spatial correlation with an intensive lignification. In the control seedlings, AMADH activity or lignification in these tissues could not be visualized. Thus, we conclude that, in plants, AMADH may participate in processes of adaptation to stress events caused by mechanical injury, which involve polyamine catabolism, GABA production and lignification

High-level expression and characterization of Zea mays cytokinin oxidase/dehydrogenase in Yarrowia lipolytica

International audienc

Mechanism-based inhibitors of cytokinin oxidase/dehydrogenase attack FAD cofactor.

International audienceCytokinin oxidases/dehydrogenases (CKOs) mediate catabolic regulation of cytokinin levels in plants. Several substrate analogs containing an unsaturated side chain were studied for their possible inhibitory effect on maize CKO (ZmCKO1) by use of various bioanalytical methods. Two allenic derivatives, N(6)-(buta-2,3-dienyl)adenine (HA-8) and N(6)-(penta-2,3-dienyl)adenine (HA-1), were identified as strong mechanism-based inhibitors of the enzyme. Despite exhaustive dialysis, the enzyme remained inhibited. Conversely, substrate analogs with a triple bond in the side chain were much weaker inactivators. The crystal structures of recombinant ZmCKO1 complexed with HA-1 or HA-8 were solved to 1.95 A resolution. Together with Raman spectra of the inactivated enzyme, it was revealed that reactive imine intermediates generated by oxidation of the allenic inhibitors covalently bind to the flavin adenine dinucleotide (FAD) cofactor. The binding occurs at the C4a atom of the isoalloxazine ring of FAD, the planarity of which is consequently disrupted. All the compounds under study were also analyzed for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4 in a bacterial receptor assay and for cytokinin activity in the Amaranthus bioassay. HA-1 and HA-8 were found to be good receptor ligands with a significant cytokinin activity. Nevertheless, due to their ability to inactivate CKO in the desired time intervals or developmental stages, they both represent attractive compounds for physiological studies, as the inhibition mechanism of HA-1 and HA-8 is mainly FAD dependent

Phenyl- and benzylurea cytokinins as competitive inhibitors of cytokinin oxidase/dehydrogenase: a structural study

Cytokinin oxidase/dehydrogenase (CKO) is a flavoenzyme, which irreversibly degrades the plant hormones cytokinins and thereby participates in their homeostasis. Several synthetic cytokinins including urea derivatives are known CKO inhibitors but structural data explaining enzyme inhibitor interactions are lacking. Thus, an inhibitory study with numerous urea derivatives was undertaken using the maize enzyme (ZmCKO1) and the crystal structure of ZmCKO1 in a complex with N-(2-chloro-pyridin-4-yl)-N'-phenylurea (CPPU) was solved. CPPU binds in a planar conformation and competes for the same binding site with natural substrates like N(6)-(2-isopentenyl)adenine (iP) and zeatin (Z). Nitrogens at the urea backbone are hydrogen bonded to the putative active site base Asp169. Subsequently, site-directed mutagenesis of L492 and E381 residues involved in the inhibitor binding was performed. The crystal structures of L492A mutant in a complex with CPPU and N-(2-chloro-pyridin-4-yl)-N'-benzylurea (CPBU) were solved and confirm the importance of a stacking interaction between the 2-chloro-4-pyridinyl ring of the inhibitor and the isoalloxazine ring of the FAD cofactor. Amino derivatives like N-(2-amino-pyridin-4-yl)-N'-phenylurea (APPU) inhibited ZmCKO1 more efficiently than CPPU, as opposed to the inhibition of E381A/S mutants, emphasizing the importance of this residue for inhibitor binding. As highly specific CKO inhibitors without undesired side effects are of major interest for physiological studies, all studied compounds were further analyzed for cytokinin activity in the Amaranthus bioassay and for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4. By contrast to CPPU itself, APPU and several benzylureas bind only negligibly to the receptors and exhibit weak cytokinin activity

Structural and Biochemical Characterization of Aldehyde Dehydrogenase 12, the Last Enzyme of Proline Catabolism in Plants

International audienceHeterokonts, Alveolata protists, green algae from Charophyta and Chlorophyta divisions, and all Embryophyta plants possess an aldehyde dehydrogenase (ALDH) gene named ALDH12. Here, we provide a biochemical characterization of two ALDH12 family members from the lower plant Physcomitrella patens and higher plant Zea mays. We show that ALDH12 encodes an NAD +-dependent glutamate gamma-semialdehyde dehydrogenase (GSALDH), which irreversibly converts glutamate gamma-semialdehyde (GSAL), a mitochondrial intermediate of the proline and arginine catabolism, to glutamate. Sedimentation equilibrium and small-angle X-ray scattering analyses reveal that in solution both plant GSALDHs exist as equilibrium between a domain-swapped dimer and the dimer-ofdimers tetramer. Plant GSALDHs share very low-sequence identity with bacterial, fungal, and animal GSALDHs (classified as ALDH4), which are the closest related ALDH superfamily members. Nevertheless, the crystal structure of ZmALDH12 at 2.2-A resolution shows that nearly all key residues involved in the recognition of GSAL are identical to those in ALDH4, indicating a close functional relationship with ALDH4. Phylogenetic analysis suggests that the transition from ALDH4 to ALDH12 occurred during the evolution of the endosymbiotic plant ancestor, prior to the evolution of green algae and land plants. Finally, ALDH12 expression in maize and moss is downregulated in response to salt and drought stresses, possibly to maintain proline levels. Taken together, these results provide molecular insight into the biological roles of the plant ALDH12 family. (C) 2018 Elsevier Ltd. All rights reserved